Tagged ligands for enrichment of rare analytes from a mixed sample

ABSTRACT

Method of enriching specific cells from cellular samples are disclosed, comprising contacting in solution a cellular sample with affinity-tagged ligands (ATLs) each comprising a first ligand linked to an affinity tag, wherein the ligand selectively binds a cellular marker of the rare cells and the affinity tag can be selectively captured by a capture moiety, wherein the affinity tags do not comprise a magnetic particle; and flowing the sample through a microfluidic device comprising the capture moiety to selectively retain ATL-bound cells. Methods for enriching circulating tumor cells, and devices for enriching specific cells from cellular samples are also disclosed.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/103,352, filed Dec. 11, 2013 which is a continuation of U.S.application Ser. No. 13/692,710, filed Dec. 3, 2012, now abandoned,which is a continuation of U.S. application Ser. No. 13/182,003, filedJul. 13, 2011, now abandoned, which is a divisional of U.S. applicationSer. No. 12/037,077, now U.S. Pat. No. 8,008,032, filed Feb. 25, 2008,which applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 11, 2014, isnamed 32061-721-302-Seqlist.txt and is 22,363 bytes in size.

BACKGROUND OF THE INVENTION

Analysis of a complex mixture such as blood can be difficult butprovides valuable information. For example, CD4 T cell levels canreflect the course of disease in AIDS patients, cellular lymphoid andmyeloid markers can aid leukemia and lymphoma diagnoses, and thepresence of errant epithelial cells in the blood may signify ametastasizing cancer.

Such complex mixtures can be analyzed by fluorescent activated cellsorting (FACS), a technology that can quantitate marked analytes andalso separate them out from the mixtures. Another popular method of cellseparation involves magnetically labeling a target population of cells,e.g. with ferromagnetic beads, followed by sorting the labeled cells bypassing them through a receptacle positioned within a magnetic field, aprocess also known as magnetic activated cell sorting (MACS). Despitetheir widespread use, FACS and MACS technologies have disadvantages, forexample, they require machinery that is expensive and difficult tomaintain. FACS technology has the added disadvantage of having limitedportability.

Given the heavy demand, new approaches and technologies for cell sortingare needed for medical diagnostics and other applications.

SUMMARY OF THE INVENTION

In one aspect, methods for enriching specific cells in a cellular sampleare provided. The methods comprise a method for enriching rare cellsfrom a cellular sample comprising contacting in solution a cellularsample with affinity-tagged ligands (ATLs) each comprising a firstligand linked to an affinity tag, wherein the ligand selectively binds acellular marker of the rare cells and the affinity tag can beselectively captured by a capture moiety, wherein the affinity tags donot comprise a magnetic particle; and flowing the sample through amicrofluidic device comprising the capture moiety to selectively retainATL-bound cells.

In some embodiments, the method comprises a method for enriching rarecells from a cellular sample comprising: contacting in solution thecellular sample with affinity-tagged ligands (ATLs) each comprising:

a ligand linked to a plurality of affinity tags, wherein the ligandselectively binds a cellular marker of the rare cells and the affinitytag can be selectively captured by a capture moiety and wherein theratio of ligand:affinity tag of the ATL's is less than 1:1; flowing thesample through a microfluidic device comprising the capture moiety toselectively retain ATL-bound cells.

In one aspect, methods for enriching specific cells in a cellular sampleare provided. The methods comprise a method for enriching rare cells,wherein the rare cells are circulating tumor cells or epithelial cells,from a cellular sample comprising contacting in solution a cellularsample with affinity-tagged ligands (ATLs) each comprising a firstligand linked to an affinity tag, wherein the ligand is folic acid,Dolichos Biflorus Agglutinin (DBAs), epidermal growth factor (EGF), EGFpeptide (amino acid sequence YHWYGYTPQNVI, SEQ ID NO:1), epi peptide(amino acid sequence QMARIPKRLARH, SEQ ID NO:2), transforming growthfactor-alpha (TGF-alpha), Urokinase Plasminogen Activator (UPA), FasL,MUC1/sec, catenin, ICAM-1, plasminogen, or the like and selectivelybinds a cellular marker of the rare cells and the affinity tag can beselectively captured by a capture moiety, wherein the affinity tagscomprise biotin, desthiobiotin, histidine, polyhistidine, myc,hemagglutinin (HA), FLAG, fluorescence tag, tandem affinity purification(TAP) tags, FLAG, or glutathione S transferase (GST) or derivativesthereof; and flowing the sample through a microfluidic device comprisingthe capture moiety to selectively retain ATL-bound cells; wherein thecapture moiety comprises avidin, streptavidin, Neutravidin™, nickel, orglutathione or other molecule capable of binding the affinity tag; andwherein the cellular marker is a cancer marker for one or more ofbreast, prostate, liver, ovary, skin, colon, rectum, cervix, esophagus,stomach, brain, lung, or endometrium cancer.

In some embodiments, the method comprises a method for enriching rarecells from a cellular sample comprising: contacting in solution thecellular sample with affinity-tagged ligands (ATLs) each comprising:

a ligand linked to a plurality of affinity tags, wherein the ligand isfolic acid or epidermal growth factor (EGF) and. selectively binds acellular marker of the rare cells wherein the rare cells are circulatingtumor cells or epithelial cells, and the affinity tag and wherein theratio of ligand:affinity tag of the ATL's is less than 1:1; flowing thesample through a microfluidic device comprising the capture moiety toselectively retain ATL-bound cells.

In one aspect, methods for enriching specific cells in a cellular sampleare provided. The methods comprise a method for enriching rare cells,wherein the rare cells are circulating tumor cells or epithelial cells,from a cellular sample comprising contacting in solution a cellularsample with affinity-tagged ligands (ATLs) each comprising a firstligand linked to an affinity tag, wherein the ligand is an antibody thatis anti-EpCam; anti-E-Cadherin, anti-Mucin-1, anti-Cytokeratin (CK) 8,anti-epidermal growth factor receptor (EGFR), anti-cytokeratin (CK)19,anti-ErbB2, anti-PDGF, anti-L6, or anti-leukocyte associated receptor(LAR) and selectively binds a cellular marker of the rare cells and theaffinity tag can be selectively captured by a capture moiety, whereinthe affinity tags comprise biotin, desthiobiotin, histidine,polyhistidine, myc, hemagglutinin (HA), FLAG, fluorescence tag, tandemaffinity purification (TAP) tags, FLAG, or glutathione S transferase(GST) or derivatives thereof; and flowing the sample through amicrofluidic device comprising the capture moiety to selectively retainATL-bound cells; wherein the capture moiety comprises avidin,streptavidin, Neutravidin™, nickel, or glutathione or other moleculecapable of binding the affinity tag; and wherein the cellular marker isa cancer marker for one or more of breast, prostate, liver, ovary, skin,colon, rectum, cervix, esophagus, stomach, brain, lung, or endometriumcancer.

In some embodiments, the method comprises a method for enriching rarecells from a cellular sample comprising: contacting in solution thecellular sample with affinity-tagged ligands (ATLs) each comprising:

a ligand linked to a plurality of affinity tags, wherein the ligand isan antibody that is anti-EpCam; anti-E-Cadherin, anti-Mucin-1,anti-Cytokeratin (CK) 8, anti-epidermal growth factor receptor (EGFR),anti-cytokeratin (CK)19, anti-ErbB2, anti-PDGF, anti-L6, oranti-leukocyte associated receptor (LAR) and. selectively binds acellular marker of the rare cells wherein the rare cells are circulatingtumor cells or epithelial cells, and the affinity tag and wherein theratio of ligand:affinity tag of the ATL's is less than 1:1; flowing thesample through a microfluidic device comprising the capture moiety toselectively retain ATL-bound cells.

In some embodiments, the method comprises a method for enriching rareanalytes from a sample comprising:

contacting in solution said sample with a plurality of affinity-taggedligands (ATLs) wherein said mixture of ATL's comprises: a first ATLcomprising a first ligand that selectively binds a first marker of rareanalytes, wherein the first ligand is linked to a first affinity tagthat is selectively captured by a first capture moiety; and a second ATLcomprising a second ligand that selectively binds a second marker ofrare analytes, wherein said second ligand is linked to a second affinitytag, wherein the second affinity tag is selectively captured by thefirst capture moiety; and contacting the sample with the capture moietyto selectively enrich the rare analytes.

In some embodiments, the method comprises a method for enriching rareanalytes from a sample comprising contacting in solution said samplewith a plurality of affinity-tagged ligands (ATLs) wherein said mixtureof ATL's comprises: a first ATL comprising a first ligand thatselectively binds a first marker of rare analytes, wherein the firstligand is linked to a first affinity tag that is selectively captured bya first capture moiety; and a second ATL comprising a second ligand thatselectively binds a second marker of rare analytes, wherein said secondligand is linked to a second affinity tag, wherein the second affinitytag is selectively captured by the first capture moiety; contacting thesample with the capture moiety to selectively enrich the rare analytes;wherein the first affinity tag and said second affinity tag areidentical; and wherein the ligand is folic acid, (DBA), EGF; EGF peptide(amino acid sequence YHWYGYTPQNVI, SEQ ID NO:1), epi peptide (amino acidsequence QMARIPKRLARH, SEQ ID NO:2), TGF-alpha; Urokinase PlasminogenActivator (UPA), FasL, MUC1/sec, catenin, ICAM-1, plasminogen, or thelike, or wherein the ligand is an antibody that is anti-EpCam;anti-E-Cadherin, anti-Mucin-1, anti-Cytokeratin (CK) 8, anti-epidermalgrowth factor receptor (EGFR), anti-cytokeratin (CK)19, anti-ErbB2,anti-PDGF, anti-L6, or anti-leukocyte associated receptor (LAR).

In some embodiments, the method comprises a method for enriching rareanalytes such as cells from a sample such as a blood sample comprising:contacting in solution said sample with a plurality of affinity-taggedligands (ATLs) wherein said mixture of ATL's comprises: a first ATLcomprising a first ligand that selectively binds a first marker of rareanalytes, wherein the first ligand is linked to a first affinity tagthat is selectively captured by a first capture moiety; and a second ATLcomprising a second ligand that selectively binds a second marker ofrare analytes, wherein said second ligand is linked to a second affinitytag, wherein the second affinity tag is selectively captured by thefirst capture moiety; contacting the sample with the capture moiety toselectively enrich the rare analytes; wherein the mixture of ATL'scomprises at least 3, 4, 5, 6, 7, 8, 9, or 10 ATL's each of whichcomprises an affinity tag that can be selectively captured by the firstcapture moiety;

In some embodiments, the method comprises a method for enriching rareanalytes such as cells from a sample such as a blood sample comprising:contacting in solution said sample with a plurality of affinity-taggedligands (ATLs) wherein said mixture of ATL's comprises: a first ATLcomprising a first ligand that selectively binds a first marker of rareanalytes, wherein the first ligand is linked to a first affinity tagthat is selectively captured by a first capture moiety; and a second ATLcomprising a second ligand that selectively binds a second marker ofrare analytes, wherein said second ligand is linked to a second affinitytag, wherein the second affinity tag is selectively captured by thefirst capture moiety; contacting the sample with the capture moiety toselectively enrich the rare analytes; wherein the mixture of ATL'scomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ATL's each of whichcomprises an affinity tag that can be selectively captured by the firstcapture moiety and wherein each of the ATL's comprises a ratio ofLigand:Affinity Tag that is less than 1:5; and wherein the capturemoiety is in a microfluidic device such as a microfluidic devicecomprising an array of obstacles; wherein the first or second ligand isfolic acid, (DBA), EGF; EGF peptide (amino acid sequence YHWYGYTPQNVI,SEQ ID NO:1), epi peptide (amino acid sequence QMARIPKRLARH, SEQ IDNO:2), TGF-alpha; Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec,catenin, ICAM-1, plasminogen, or the like, or wherein the ligand is anantibody that is anti-EpCam; anti-E-Cadherin, anti-Mucin-1,anti-Cytokeratin (CK) 8, anti-epidermal growth factor receptor (EGFR),anti-cytokeratin (CK)19, anti-ErbB2, anti-PDGF, anti-L6, oranti-leukocyte associated receptor (LAR); and wherein the affinity tagcomprises biotin, desthiobiotin, histidine, polyhistidine, myc,hemagglutinin (HA), FLAG, fluorescence tag, tandem affinity purification(TAP) tags, FLAG, or glutathione S transferase (GST) or derivativesthereof; wherein the ligand is linked to the affinity tag eitherdirectly or via a linker comprising modified dextran, polyethyleneglycol, polypropylene glycol, polyvinyl alcohol, orpolyvinylpyrrolidone; and wherein the capture moiety comprises avidin,streptavidin, Neutravidin™ nickel, or glutathione or other moleculecapable of binding the affinity tag.

In some embodiments, the method comprises a method of diagnosing acondition such as cancer in a patient comprising: enriching rare cellsfrom the patient through any of the methods described herein; andevaluating, identifying, or quantitating the ATL bound cells.

In some embodiments, the invention comprises a reagent comprising aplurality of affinity tagged ligands each of which comprises a ligandthat selectively binds a different cancer cellular marker linked to anidentical affinity tag that is selectively captured by a capture moietywherein the ratio of ligand:affinity tag per ATL is less than 1:1;wherein the reagent comprises at least 1, 2, 3, 4, or 5 different ATL's.

In some embodiments, the device comprises a microfluidic devicecomprising an array of obstacles, wherein the obstacles arefunctionalized with capture moieties comprising EGF-peptide,EPI-peptide, folate or DBA.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts two fluorescent images illustrating the capture of lungcancer cells with a non-specific affinity tagged ligand (biotinylatedanti-IgG).

FIG. 1B depicts two fluorescent images illustrating the capture of lungcancer cells with biotinylated anti-EpCam antibody, an antibody that isspecific for an epithelial cell marker present on lung cancer cells.

FIG. 2A is a diagram illustrating the capture of cells with a ligandtagged with an affinity tag such as biotin, histidine, or a magneticparticle (top panel), and the affinity-based capture of such cells usinga microfluidic chip coated with a capture moiety capable of interactingwith the affinity tag (bottom panel).

FIG. 2B is a fluorescent image illustrating the capture of cells boundto Biotin-tagged EGF using a microfluidic device coated with Avidin, acapture moiety capable of interacting with the affinity tag.

FIG. 3 is a Table of exemplary markers contemplated herein.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions, methods and devices forthe detection and isolation of rare analytes from a mixed sample. Rareanalytes include cells and particles, such as eukaryotic cells,prokaryotic cells, cellular organelles, cellular fragments, viruses,nucleic acids, proteins, and protein complexes. A mixed sample mayinclude a fluid sample, e.g., water, air, bodily fluids such as blood,urine, etc. A mixed sample may also include a solid sample that isliquefied, e.g., a tumor biopsy, food solids, etc.

Enrichment, detection, and isolation of rare analytes is accomplishedusing affinity-tagged ligands (ATLs). An ATL may comprise Formula I:L _(x−)AT_(n),  (Formula I)wherein:L is a ligand that selectively binds a marker for a rare analyte;x is the number of ligands within each ATL;AT is an affinity tag which can be selectively captured using a capturemoiety; andn is the number of ATs linked to the ligand.

L and AT may be linked together directly or indirectly (via a linker).Linkers may be straight or branched. A linker may be a peptide,polypeptide, protein, and the like. In some embodiments, the linkermoiety is a branched polymer. Such branched polymers include modifieddextran, polyethylene glycol, polypropylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, polyethylene acrylamide and combinations thereof.In some embodiments, the linker is branched and comprises multiple ATs.For example, in some embodiments an ATL comprises at least 5, 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more ATs per L. When asingle ligand is linked to multiple AT's, the AT's can each be linked tothe ligand via a single linker or via a plurality of linkers (e.g., onelinker per AT).

Examples of ligands contemplated in the methods herein include ligandsthat selectively bind cell surface marker(s) (e.g., receptors andadhesion molecules) of rare cells of interest. A ligand can comprisemolecule(s), peptide(s), polypeptides, particles, and the like, and/orfragments thereof. The ligand can be naturally occurring or artificiallysynthesized. Rare cells include circulating tumor cells (CTCs),circulating epithelial cells or circulating stem cells.

In some embodiments, a ligand comprises an antibody, such as amonoclonal antibody or a polyclonal antibody. Examples of ligandantibodies contemplated herein include antibodies that selectively bindCTCs, circulating epithelial cells, or circulating stem cells. In someembodiments, such antibodies selectively bind an antigen such as EpCam;E-Cadherin, Mucin-1, Cytokeratin (CK) 8, epidermal growth factorreceptor (EGFR), cytokeratin (CK)19, ErbB2, PDGF, L6, or leukocyteassociated receptor (LAR). In certain embodiments, the ligand may be anantibody to a marker listed in FIG. 3.

In other embodiments, a ligand is not an antibody. Examples ofnon-antibody ligands include folic acid, Dolichos Biflorus Agglutinin(DBA), epidermal growth factor (EGF), EGF peptide (amino acid sequenceYHWYGYTPQNVI, SEQ ID NO:1), epi peptide (amino acid sequenceQMARIPKRLARH, SEQ ID NO:2), transforming growth factor-alpha(TGF-alpha), Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec,catenin, ICAM-1, plasminogen, or the like. In some embodiments, theligand activates a cellular process, and in other embodiments, theligand may not activate a cellular process.

An ATL may have a single ligand or multiple ligands (e.g., x>1, or x>2,5, 10, 20, or more). When an ATL comprises multiple ligands, each ligandmay be identical to the other ligands, different from the other ligands,or a combination thereof.

Affinity tags (AT) can include molecules, peptides, polypeptides,particles, and/or other substances capable of being captured by acapture moiety or enrichment device. Examples of ATs include biotin,desthiobiotin, histidine, poly-histidine, glutathione S transferase(GST), myc, hemagglutinin (HA), FLAG, fluorescence tag, tandem affinitypurification (TAP) tags, or derivatives thereof. The TAP tag maycomprise calmodulin-binding protein, TEV, and protein A or G subunit. Insome embodiments, the affinity tag comprises a magnetic particle. Inother embodiments, the affinity tag is a molecule or particle other thana magnetic particle. In some instances, an affinity tag does notcomprise an antibody.

As described above, an ATL may comprise one AT, or, for example, maycomprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or moreATs.

To detect rare analytes (or single analyte) in a fluid sample, thesample is mixed with a homogenous or heterogeneous set of ATLs. Thesample is incubated with the ATLs for a sufficient amount of time toenable binding of the ligands to the receptors or rare analytes. Thesample and ATLs are then applied to one or more capture moieties thatselectively bind the affinity tags, thereby allowing enrichment of thebound rare analytes. The capture moiety can be coupled to a solidsupport (e.g. a tube, a flat substrate, or an array of obstacles).

To detect rare analytes in a solid or semi-solid sample, the sample isfirst solubilized and then mixed with the mixture of ATLs as describedabove. Once the sample is incubated with the ATLs for a sufficientperiod of time to allow binding between the ligands and the rare analytemarkers, the sample is applied to one or more capture moieties thatselectively binds to the affinity tag.

Capture moieties may be inorganic molecules, organic molecules,peptides, polypeptides, and/or antibodies, and the like, or may includefragments of, or derivatives, of same. The capture moieties are capableof binding to, or interacting with, an affinity tag. Examples of capturemoieties include, but are not limited to, a magnet or magnetizedsubstrate, avidin, streptavidin, Neutravidin™, glutathione, nickel,calcium/calmodulin, or IgG. Other examples of capture moieties includemagnetic or magnetized surfaces, avidin and avidin-coated particles.

In some instances, capture moieties are part of a microfluidic device(e.g., a microfluidic gap or channel coated with capture moieties). Amicrofluidic device contemplated herein can comprise an array ofobstacles that form a network of microfluidic channels.

The affinity tags can bind irreversibly or reversibly to the capturemoiety(ies). In some instances, an affinity tag may be releasable fromthe capture moiety. For example, desthio-biotin may be used as anaffinity tag with avidin as the capture moiety. The bond between biotinand avidin can then be released by applying desphio-biotin or biotin tothe sample.

In one example, the ATL's herein can be used to enrich rare analytessuch as cells from a mixed sample such as blood. Such method comprisesthe step of contacting in solution a test sample with affinity-taggedligands (ATLs). Each ATL comprises: at least one ligand linked to atleast one affinity tag. The ligand and affinity tag are optionally likedvia a linker comprising one or more of modified dextran, polyethyleneglycol, polypropylene glycol, polyvinyl alcohol, orpolyvinylpyrrolidone.

The ligands of the ATL's selectively bind a one or more markers of therare analytes. The affinity tags of the ATLs are ones that can beselectively captured by a single capture moiety or a plurality ofcapture moieties. Examples of capture moieties contemplated for any ofthe embodiments herein include avidin, streptavidin, Neutravidin™,nickel, or glutathione. The affinity tags may be one that does notcomprise a magnetic particle. Examples of affinity tags include biotin,desthiobiotin, histidine, polyhistidine, myc, hemagglutinin (HA), FLAG,fluorescence tag, tandem affinity purification (TAP) tags, FLAG,glutathione S transferase (GST) or derivatives thereof. After incubationfor a sufficient amount of time to allow the sample and ATL's to mix,the mixed sample is contacted with one or more capture moieties or isflowed through a microfluidic device comprising the one or more capturemoieties. This selectively retains ATL-bound analytes or cells.

In some instances, each ATL comprises a ratio of ligand to affinity tagsuch that there are more affinity tags for each ligand. In someinstances the ligand:affinity tag ratio is less than 1:1 or less than1:5 or less than 1:10 or less than 1:100. Furthermore, a single ATL canhave at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different ligands. Each ofthe ligands can be specific to a different marker, such as a cancermarker.

Cellular cancer markers contemplated herein include markers for a cancersuch as breast, prostate, liver, ovary, skin, colon, rectum, cervix,esophagus, stomach, brain, lung, pancreatic, or endometrium cancer. Insome instances a cellular marker is epidermal growth factor receptor(EGFR), EPCAM, or folic acid receptor. In some instances, the cellularmarker is E-Cadherin, Mucin-1, Cytokeratin (CK) 8, cytokeratin (CK)19,ErbB2, PDGF, L6, or leukocyte associated receptor (LAR) In otherinstances it is any marker listed in the table of FIG. 3.

To enrich rare analytes such as rare cells (epithelial cells or CTC)found in bodily fluid (e.g., blood), the ligands in the ATL's can be oneor more of the following: folic acid, epidermal growth factor (EGF),transforming growth factor-alpha (TGF-alpha), Urokinase PlasminogenActivator (UPA), FasL, MUC1/sec, catenin, ICAM-1, plasminogen, orfragments thereof. In some embodiments, the ligands comprise haptens. Insome instances, the ligand is an antibody and in others it is not anantibody.

In some instances, a reagent used to enrich/detect rare analytescomprises a plurality of affinity-tagged ligands (ATLs) wherein each ATLcomprises: a first ATL comprising a first ligand that selectively bindsa first marker of the rare analytes linked to a first affinity tag thatis selectively captured by a first capture moiety; and a second ATLcomprising a second ligand that selectively binds a second marker linkedto a second affinity tag, wherein the second affinity tag is selectivelycaptured by the first capture moiety. In such embodiments, the reagentdescribed above is mixed with a mixed sample of analytes. After allowingthe ATL's and sample to mix for a sufficient amount of time for bindingto occur, the sample/ATL mixture is contacted with the first capturemoiety to selectively enrich the rare analytes. The capture moiety canbe avidin and the capture moiety can be in a microfluidic gap orchannel. The first and second affinity tags can be identical (bothbiotin) or different (biotin and desphio-biotin) but both selectivelycaptured by the same capture moiety. In some instances, a mixed samplesuch as the one described above is contacted with at least 3, 4, 5, 6,7, 8, 9, or 10 different ATL's each of which comprises an affinity tagthat can be selectively captured by a single capture moiety or the sameaffinity tag.

A sample, such as a blood sample, can be mixed with one or moredifferent ATLs. In certain embodiments, ATLs of similar configurationmay be contacted with the targeted analytes. In other embodiments, amixture of different ATLs may be used. In still other embodiments,step-wise purification of the analyte may occur through sequential useof the same or different ATLs.

An exemplary ATL can have a single ligand comprising anti-EpCAM antibodylinked to a plurality of ATs, each of which comprises a biotin molecule.The ATs are linked to the ligand via a branched linker such that eachligand is coupled to at least five ATs. In some embodiments, the ligandmay bind the marker (EpCAM) at more than one site. In other embodiments,e.g., EGF, the ligand binds the marker only once. Once the sample ismixed with the ATLs, the sample flows through a microfluidic device,optionally comprising an array of obstacles, coated with avidin capturemoieties. Enriched rare cells (e.g., circulating epithelial cells) canthen be visualized/detected using any means known in the art, includingfluorescent in situ hybridization (FISH) staining.

Some applications may require analysis downstream of the capture of theATL-bound cells. In some embodiments, the ATLs may be conjugated to oneor more detectable labels. Such detectable labels comprise fluorescentmarkers, HRP-alkaline phosphatase detection or quantum dots. In someembodiments, in addition to an ATL, the cells may be contacted with adetectably-labeled ligand specific for a cellular marker. Suchdetectable labels may comprise one or more of fluorescent probes,magnetic particles, or quantum dots. The detectably-labeled ligandscomprise the ligands described herein relating to ATLs. An example of adetectably-labeled ligand is a fluorescently-labeled antibody.

Varying the number of affinity tags and/or ligands in a single ATL mayaffect the ability to capture target analytes. Thus, the disclosureprovides ATLs with more than one affinity tag and/or more than oneligand. The affinity tags may be identical tags, similar tags, ordifferent tags. Since higher affinity-tag-to-ligand ratios may result inenhanced or more efficient capture of cells, the ratio of affinity tagto ligand in a single ATL may be greater than or equal to 1, forexample, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, or greater.

In some embodiments, the marker is a marker for one or more of thefollowing: cancer of the breast, lung, prostate, liver, ovary, skin,colon, rectum, cervix, esophagus, stomach, brain, bladder, kidney,testicular, or endometrium. In some embodiments, the marker is for ablood cancer such as leukemia or lymphoma.

In some embodiments, vascular disease is monitored by enriching forcells with endothelial markers. Other cancers may also be monitored byenriching for endothelial cells. In some embodiments, fetal markers(e.g. Y chromosome proteins, gamma globin) are used for capturing fetalcells in the maternal circulation. In some embodiments, stem cellmarkers (CD44) are used to capture stem cells.

In certain embodiments, the methods provided comprise contacting acellular sample with one or more ATLs to enable binding to one or morecellular markers, followed by capture of the bound cells by anenrichment device. The contacting of the cellular sample with one ormore ATLs may occur under conditions pre-determined to maximize bindingefficiency. In some embodiments, the conditions may be optimized tominimize non-specific binding. For example, the timing of the contactingbetween the cellular sample and the one or more ATLs may be optimized.Such optimized or predetermined amount of time may be used for thecontacting step. Likewise, conditions for the temperature of thecontacting step may be optimized to maximize binding efficiency and/orto minimize internalization of cellular markers. A pre-determined oroptimized temperature or temperatures may be used for the contactingstep. In some embodiments, the composition of the solution containingthe cellular sample and/or the ATL may be optimized for more efficientbinding. Blocking reagents or serums may be added to aid in bindingefficiency. Examples of such blocking reagents include bovine serumalbumin (BSA), fetal calf serum (FCS), milk, and othercommercially-obtainable reagents.

The methods may further comprise a wash step in which unbound (or free)ATL is removed from the cellular sample prior to capture by theenrichment device. For example, the wash step could be accomplished byone or more rounds comprising centrifugation of the cellular sample,aspiration of the supernatant, and re-suspension of the cellular samplein wash buffer. The composition of the wash buffer may comprise bufferedsolutions and may be optimized to maximize binding efficiency. Theoptimized wash buffer may comprise one or more blocking reagents orserums described herein.

In some embodiments, the cells are added to an enrichment device thatcomprises a surface functionalized with a capture moiety recognizing theaffinity tag or a cellular marker, such as a receptor. In certainembodiments, a surface of the enrichment device is coated or treatedwith one or more capture moieties (e.g., avidin or Neutravidin) in orderto allow capture of the ATL-bound cells. In certain embodiments, suchcapture-moieties are not antibodies. In some embodiments, such capturemoieties are ligands. In some embodiments, the enrichment devicecomprises a surface coated or functionalized with a ligand that is usedto directly enrich cells expressing a specific marker from a mixed cellpopulation. In some embodiments, the surface of such enrichment deviceis functionalized with folate in order to enrich cells expressing thefolate receptor. In some embodiments, the enrichment device isfunctionalized with Dolichos Biflorus Agglutinin (DBA), epidermal growthfactor (EGF), EGF peptide (amino acid sequence YHWYGYTPQNVI, SEQ IDNO:1), epi peptide (amino acid sequence QMARIPKRLARH, SEQ ID NO:2),transforming growth factor-alpha (TGF-alpha), Urokinase PlasminogenActivator (UPA), FasL, MUC1/sec, catenin, ICAM-1, plasminogen, or thelike. Parameters such as timing, temperature, and/or buffer compositionmay be optimized to promote optimal capture by the enrichment device.

The enrichment device may include one or more arrays of obstacles thatallow lateral displacement of cells and other components of fluids. Sucharrays of obstacles comprise different configurations. In oneconfiguration, a row or rows of obstacles are arranged with a 50% offsetfrom other randomly- or non-randomly-arranged obstacles creatingrestricted and expanded gaps.

In some embodiments, the obstacles within the array of obstacles arecoated or treated with one or more capture moieties or ligands. In someembodiments, the enrichment device is capable of sorting cells based onanother variable, e.g. hydrodynamic size, as well as capturing ATL-boundcells. Enrichment devices include, but are not limited to centrifuge,microfuge and ultracentrifuge test tubes. In some embodiments, theenrichment device is a column or other such receptacle such as amagnetic activated cell sorting (MACS) column.

Any of the ATLs herein may be designed to enrich cancer cells as well asprognose and diagnose cancer conditions. For example, cancer can bestaged or diagnosed by enriching rare cells (e.g., epithelial cells,circulating cancer stem cells) using ATL's by any of the means describedabove. Subsequently, the enriched cells can be further evaluated orquantified. In one instance, diagnosis or prognoses comprises comparingthe percentage or quantity of epithelial cells in the blood of a testsubject with that in the blood of a healthy control subject, a panel ofhealthy control subjects, known standard levels, or similar informationobtained from the same patient at a different point in time.

Peptide Library

In some embodiments, the present disclosure provides a method forscreening a combinatorial peptide library for peptide ligands capable ofbinding cell surface markers. Such methods comprise contacting a sampleof cancer cells with a combinatorial peptide library, wherein thepeptides have been pre-labeled with one or more affinity tags, describedherein; purifying out the cells selectively binding to candidate peptideligands; and identifying the candidate ligands bound to the cells. Incertain embodiments, the purifying may comprise the lateral flow of thecellular sample through a device coated with capture moieties thatselectively bind the ATs so as to permit capture of the ATL bound cells.

EXAMPLES Example 1: Capture of H1650 Lung Cancer Cells with BiotinylatedAnti-EpCam Antibody

The example depicted in FIGS. 1A & 1B illustrates a method and devicefor isolating cancer cells. The affinity-tagged ligand (ATL) in thisexample comprised biotin as the affinity tag. The ligand wasanti-Epithelial Cell Adhesion Molecule (EpCam) antibody which recognizesan epithelial cellular marker. For the experiment, H1650 lung cancercells were incubated with biotinylated goat anti-EpCam antibody for 1hour on ice (FIG. 1B). Approximately 1000 cells were input into eachexperiment. The left and right images represent replicate experiments.As a negative control, H1650 lung cancer cells were incubated withbiotinylated goat IgG antibody under the same or similar conditions(FIG. 1A). Following the incubation, the cells were washed and runthrough an array of obstacles [comprising a restricted gap—Chip 7,please confirm] chip coated with Neutravidin™. Cells were then fixed andvisualized by fluorescence microscopy.

The results of this experiment demonstrated that it is possible to useATLs to isolate cancer cells. In this example, between 20 and 30% of thetotal H1650 lung cancer cell population were recovered when anti-EpCamantibody was used as the ligand. To calculate the percentage (%), the #of cells captured was divided by the total # of cells passed onto thechip, and the resulting value was multiplied by 100. In contrast, only7-8% of cells were recovered when the goat IgG was used.

Example 2: Capture of Cells after Solution Phase Binding of the Cellswith an Affinity-Tagged Ligand

The examples depicted in FIGS. 2A & 2B illustrate the capture of cellslabeled with an affinity tag before being subjected to a microfluidicdevice. FIG. 2A is a diagram depicting a cell being contacted with anaffinity-tagged ligand in solution (top panel) and the capture of suchcell with a microfluidic device coated with a capture moiety capable ofrecognizing the affinity-tag (lower panel).

FIG. 2B illustrates the capture of cells bound to EGF peptide taggedwith biotin. In this example, MDA-MB-231 cells (a breast cell line whichexpresses low levels of EpCam) were contacted with biotin-tagged EGFpeptide in solution under conditions favorable for binding. The cellswere then subjected to a chip coated with neutravidin, a capture moietycapable of recognizing the affinity tag. Following fixation, thecaptured cells were visualized by fluorescent microscopy. The resultsdemonstrate significant capture of cells using this method.

The invention claimed is:
 1. A method for enriching rare analytes from asample, comprising: a) contacting said sample with an affinity-taggedligand (ATL) complex wherein said ATL complex comprises: i) an affinitytag comprising a magnetic particle; and ii) at least a first ligand anda second ligand different from said first ligand, wherein said firstligand and said second ligand are linked to said affinity tag; andwherein said first ligand and said second ligand selectively binddifferent markers of said rare analytes, thereby labeling said rareanalytes with said ATL complex; b) flowing said sample comprising saidATL complex-labeled rare analytes through a microfluidic enrichmentdevice comprising an array of obstacles, wherein said obstacles arearranged in rows offset from other obstacles to create restricted andexpanded gaps and to thereby laterally displace ATL complex-labeled rareanalytes based on their hydrodynamic size, and wherein at least some ofsaid obstacles are coated or treated with one or more capture moietiesthat capture the labeled rare analytes; wherein said capture moietiescomprise a magnet or magnetized substrate that interacts with themagnetic particle of the affinity tag, to selectively enrich saidlabeled rare analytes.
 2. The method of claim 1, wherein contacting saidsample with an ATL complex is performed in solution.
 3. The method ofclaim 1, wherein said affinity tag is linked to at least one of saidfirst ligand and said second ligand via one or more linkers.
 4. Themethod of claim 3, wherein said one or more linkers comprise at leastone of: modified dextran; polyethylene glycol; polypropylene glycol;polyvinyl alcohol; polyvinylpyrrolidone; and polyethylene acrylamide. 5.The method of claim 1, wherein said first ligand comprises a firstantibody and said second ligand comprises a second antibody differentfrom said first antibody.
 6. The method of claim 5, wherein said firstantibody binds to a first marker for said rare analytes and said secondantibody binds to a second marker of said rare analytes, and whereinsaid first marker is different from said second marker.
 7. The method ofclaim 5, wherein at least one of said first antibody and said secondantibody is an Epithelial Cell Adhesion Molecule (EpCAM) antibody. 8.The method of claim 1, wherein said one or more markers comprise one ormore cancer markers.
 9. The method of claim 1, wherein said rareanalytes comprise rare cells.
 10. The method of claim 9, wherein saidrare cells comprise circulating tumor cells.
 11. The method of claim 1,wherein said sample comprises blood.
 12. The method of claim 11, whereinsaid rare analytes comprise rare cells.
 13. The method of claim 1,wherein said affinity tag reversibly binds to said capture moieties. 14.A method for enriching leukocytes or stem cells in a sample comprising:a. contacting said sample with an affinity-tagged ligand (ATL) complex,wherein said ATL complex comprises: i. an affinity tag comprising amagnetic particle; and ii. at least a first ligand and a second liganddifferent from said first ligand, wherein said first ligand and saidsecond ligand are linked to said affinity tag, and wherein said firstligand and said second ligand selectively bind different markers of saidleukocytes or stem cells, thereby labeling said leukocytes or stem cellswith said ATL complex; b. flowing said sample comprising said ATLcomplex-labeled leukocytes or stem cells through a microfluidic devicecomprising an array of obstacles, wherein said obstacles are arranged inrows offset from other obstacles and that laterally displace the ATLcomplex-labeled leukocytes or stem cells based on their hydrodynamicsize; and c. flowing said sample comprising said ATL complex-labeledspecific cells that have been flowed through the microfluidic device instep b) through an enrichment device comprising a surface functionalizedwith a capture moiety, wherein said capture moiety comprises a magnet ormagnetized substrate that interacts with the magnetic particle of theaffinity tag on the ATL complex-labeled leukocytes or stem cells toselectively enrich said ATL complex-labeled specific cells.
 15. Themethod of claim 14 wherein at least one ligand is an antibody.
 16. Themethod of claim 15 wherein said antibody is an anti-leukocyte associatedreceptor antibody.
 17. The method of claim 15 wherein at least one ofsaid antibodies is an anti-leukocyte associated receptor antibody. 18.The method of claim 14, wherein both the first ligand and said secondligand are antibodies.
 19. The method of claim 14, wherein said affinitytag is linked to at least one of said first ligand and said secondligand via one or more linkers.
 20. The method of claim 19, wherein saidone or more linkers comprise at least one of modified dextran,polyethylene glycol, polypropylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, and polyethylene acrylamide.